Project Summary The gastrointestinal (GI) tract comprises the largest environmental interface of the body; its immune system is posed with the unique challenge of maintaining tolerance to dietary and microbial antigens while remaining poised to protect against pathogen invasion. Coordinated resistance and tolerance mechanisms serve to prevent pathogenic dissemination, limit excessive GI damage, and initiate recovery responses induced by pathogenic burden or injury. The GI tract hosts as many neurons (enteric-associated neurons, EANs) as the spinal cord and more immune cells than all other compartments together. EANs include sensory neurons, interneurons, and motor neurons with cell bodies within (intrinsic) or outside the intestine (extrinsic), which control a variety of functions within the GI tract. EANs are often targeted by enteric pathogens, resulting in functional gastrointestinal disorders post pathogen clearance. The clinical presentations of post-infectious enteric neuronal damage include unresolved low-grade intestinal inflammation, gastrointestinal motility impairment, and nerve damage. Nevertheless, the underlying mechanisms involved in infection?induced neuronal damage are incompletely understood. Our recent data indicates that murine enteric infection results in a rapid and persistent loss of iEANs, which is associated with prolonged gastrointestinal changes including intestinal dysmotility. However, infection history and microbiota composition can prevent iEAN loss or accelerate iEAN recovery, respectively; findings that may lead to a better understanding of human post-infectious IBS and additional disorders associated with EAN damage during inflammation. Imaging analyses suggested a subtype?specific neuronal loss upon Salmonella infection, and transcriptomics and genetic approaches indicated an iEAN cell death mechanism that is dependent on components of the inflammasome pathway. Depletion of intestinal muscularis macrophages (MMs), located in close proximity to enteric neurons, as well as targeting of ?2-AR on myeloid cells, resulted in enhanced infection-induced neuronal loss, suggesting a functional role for a MM tissue protective program induced upon infection. Our observations suggest a functional role for neuron?macrophage interactions in limiting infection-induced neuronal damage or accelerating neuronal recovery, supporting the significance and impact of this proposal. We will characterize mechanisms underlying neuronal cell death post enteric infection with different pathogens (Aim 1). We will also to define how microbiota manipulations can rescue neuronal death post infection, possibly defining a role for specific bacterial species in this process (Aim 2). Finally, we will investigate the cellular and molecular immune mechanisms regulating neuronal loss during heterologous secondary infections (Aim3). By utilizing imaging, cell sorting?independent transcriptomics, single-cell approaches and genetic gain? and loss?of-function approaches, this proposal aims to characterize cellular and molecular components of neuro-immune crosstalk following enteric infections.